Vaccination against variola virus (the causative agent of smallpox) is currently accomplished by vaccinia virus. Little is known about 1) the antigens and epitopes targeted by the cellular responses in humans immunized with vaccinia virus, and 2) which responses are crossreactive with variola virus and hence would be expected to contribute to the protection engendered by the vaccine. In the first part of the studies proposed herein, we will 1) determine immunodominant antigens recognized by Class I and Class II restricted responses in humans immunized with vaccinia virus, 2) map the epitopes recognized within each antigen, and 3) determine their degree of crossreactivity with homologous variola virus-derived sequences. We anticipate that these studies will lead to the definition of a broad range of epitopes, facilitate a rigorous definition of correlates of protection against smallpox infection in humans, and also enable the evaluation of the performance of different vaccine candidates. Vaccinia virus is also actively investigated as a potential vaccine delivery vehicle, either alone or in prime/boost regimens, for disease indications such as HIV, malaria and cancer. Thus, it should be noted that identification and characterization of the determinants recognized by humans infected/vaccinated by vaccinia virus would also enable the characterization and optimization of experimental vaccines utilizing vaccinia virus-derived vectors as a delivery system. The vaccinia-based vaccines currently available, while effective, are associated with significant and serious, albeit rare, side effects. Because of these side effects, and because of the worldwide eradication of variola virus, vaccination of the general population was deemed as no longer desirable. Recent renewed concerns have been raised over bioterrorist use of the virus. In the context of the studies proposed herein, a concern could be raised that if the vaccinia-induced protection is mediated by relatively few immunodominant and crossreactive antigens, a modified smallpox virus could be engineered that lacks those crossreactive epitopes. Under this terrifying scenario, the protection elicited by the vaccinia would be ineffective against the biological weapon. In the second part of the grant, we propose to counter this risk through the identification of variola virus-specific determinants derived from immunodominant antigens in the context of the vaccinia virus responses, but not crossreactive with the homologous variola virus sequences. These variola virus-derived epitopes would be incorporated in an optimized multi-determinant vaccine construct, inserted in the currently available vaccinia vaccine.